In EP 93912877.3 and U.S. Pat. No. 5,633,284 and their equivalents there is disclosed that dermatological or topical compositions comprising the combination of nitrous oxide [N2O] and at least one fatty acid, or lower alkyl ester thereof, in a dermatologically acceptable carrier medium, are useful in the treatment of a variety of skin, muscle and joint disorders. It further disclosed therein that such combinations might beneficially also include additional active ingredients. The following active ingredients are specifically mentioned in this regard: coal tar solution, collagen, nicotinamide, nicotinic acid, lanolin, vitamin E, methyl salicylate, arnica and H1-antagonist antihistamines of which only diphenhydramine chloride is specifically mentioned. In WO97/17978 and U.S. Pat. No. 6,221,377 and their corresponding applications and patents there is further disclosed that the action of analgesic, anti-inflammatory and anti-pyretic drugs may be enhanced by administering such drugs in conjunction with a medium which comprises nitrous oxide and at least one long chain fatty acid selected from the group consisting of oleic acid, linoleic acid, alpha-linolenic acid, gamma linolenic acid, arachidonic acid, and any of the C1 to C6 alkyl esters of such long chain fatty acids, mixtures of such acids and mixtures of such esters. The medium may comprise the mixture known as Vitamin F Ethyl Ester and may optionally further comprise eicosapentaenoic acid [C20:5ω3] and decosahexaenoic acid [C22:6ω3].
In WO 02/05850 there is disclosed that the effect of anti-infective drugs may be enhanced by formulation thereof in the same carrier medium.
In WO 02/05851 it is disclosed that the effect of known agents affecting the central and/or peripheral nervous system may similarly be enhanced by their formulation in the same carrier medium.
In WO 02/05849 it is disclosed that the same carrier medium may also beneficially be used for the transportation of nucleic acid compounds across cell membranes.
Antigens for use in forming vaccines are not amongst the active ingredients mentioned in the aforementioned patents and patent applications as being capable of being formulated with beneficial effect with the aid of the carrier medium therein disclosed.
The aforementioned disclosures would not have been understood as suggesting that the nitrous oxide and fatty acid combination have any adjuvant contribution in the preventative effect against diseases caused by infective agents. Within the context of the disclosure in the abovementioned patent family the notional addressee most likely would, as did the inventor, have understood the role of the anti-infective agents to be the treatment of patients already suffering from an infection.
It has now surprisingly been found that the aforesaid medium and media related thereto may itself act as an adjuvant, thereby enhancing the immunogenicity of known vaccines.
The expression “vaccine” as used herein is intended to have its extended meaning as compound(s) contributing in the prevention of infectious disease by any method or mechanism of priming of the body, and to include viral-based, peptide-based, bacterially-based, VLP-based, and synthetic compound-based formulations, but to exclude anti-infective agents used for the treatment of disease.
The exclusion of anti-infective agents from the ambit of the present invention is introduced without thereby conceding that the aforementioned patents and applications contain any disclosure of any preventative properties of such excluded compounds, or that such properties are obvious in the light of the disclosures in such patents or applications. Such inferences are specifically denied. The exclusion is introduced simply to avoid what is anticipated to be a potential obstacle to the grant of a patent in respect of a part of potential subject matter which part in itself is not considered worth contesting during examination as it might unduly delay the implementation in practice of the significant features of the present invention. It is expected that the remaining bulk of the subject matter of the present invention will greatly contribute to the accessibility of vaccines for the prevention of a large range of infections, at significantly reduced costs in cases such as Hepatitis B.
The expression “therapeutic vaccine” is further intended to cover vaccines which serve to prevent and/or treat an existing infection by eliciting and/or enhancing a specific immune response to the infective agent without the use of antimicrobial, antifungal or antiviral agents. The expression is hence intended to be understood in the wider sense of the immune response, namely all compounds that contribute in eliciting or enhancing an immune response against specific microscopic and sub microscopic organisms. This term is further specifically intended to include all antigens or native and synthetic biologicals falling within class 26 (Biologicals) of the pharmacological classification employed in the Monthly Index of Medical Specialities (“MIMS”) published by Times Media in South Africa. It is thus intended to include:
anti-bacterial vaccines;
anti-fungal vaccines;
anti-viral vaccines (including anti-retroviral vaccines);
anti-protozoal agents;
and anti spirochaete vaccines.
The finding of adjuvanticity of the media referred to above is made against the background of the fact that there appears to be no earlier suggestion in the literature to the effect that either nitrous oxide itself or the addition of nitrous oxide to the long chain fatty acids used in the formulation referred to above, has an additional stimulatory effect on the immunogenicity of vaccines.
In recent years, there has been an increasing interest in the development of novel vaccine systems for prophylactic and therapeutic purposes. Formulation strategies and the use of adjuvants that can affect the immune response in both quantitative and qualitative terms have attracted a lot of attention from those familiar with problems in drug delivery. Early efforts were focussed on parenteral vaccines and on the role of controlled release technologies with an emphasis on biodegradable microspheres1-3.
The primary aim of vaccination is to prevent disease. Historically, vaccination is the only strategy that has ever led to the elimination of a viral disease, namely smallpox. While the biology of most pathogens is less favourable than smallpox to vaccine development, some vaccines do, to varying degrees, protect humans and animals against related pathogens. An indirect relationship has been observed for vaccine immunogenicity and safety. Human immune responses to synthetic and recombinant peptide vaccines administered with standard adjuvants tend to be poor; hence there is an urgent need for effective vaccine adjuvants to enhance the immunogenicity and immunostimulatory properties of vaccines, although even imperfect vaccines could deliver public health and economic benefits and provide further insights for prevention and treatment strategies. While microbicides may usefully extend prevention options and serve as valuable prototypes for vaccine development, it is not clear that these can be delivered sustainably to everyone at risk.
Targeted vaccine campaigns against diseases such as hepatitis B have generally failed to affect disease incidence. To maximise public health and economic benefit, it may be necessary to aim for universal immunisation of children and young animals. This implies the need for an extremely high level of safety, comparable to current widely used vaccines given to children throughout the world. These considerations have favoured the use of vaccines based on relatively small parts of the pathogens.
There is of course, much greater potential in vaccines that are shown to be capable of inducing potentially relevant immune responses than in those that are not. Animal studies and laboratory measurements of human immune responses may be used to provide ‘correlates of protection’ that speed up further research and development.
Vaccines primarily use a harmless form of a pathogen, or some component of it, to induce a protective immune response involving one or both arms of the immune system: humoral and/or cell-mediated immunity. Humoral immunity is based on antibodies and the B cells that produce them. Antibodies recognise a specific target, usually a sub-part of a protein of the infective organism. ‘Neutralising’ antibodies play an important role in fighting off infections whereas cytotoxic T cells or CD8+ cells play a major role in cell-mediated immunity. Cytotoxic T cells are able to destroy most pathogen-infected cells, identified by the presence of very small fragments of pathogen proteins that are displayed on the cell surface, bound to cell proteins. Helper T cells (CD4 cells) recognise fragments of pathogens, displayed on the surface of specialised ‘antigen presenting cells (APC)’. These produce proteins, which activate B cells and/or cytotoxic T cells. When the immune system is activated by vaccination, memory T cells and sometimes memory B cells are produced. These cells enable a rapid and effective immune response when the pathogen itself is encountered, preventing infection and/or disease.
A major hindrance that has prevented the development of effective mass immunization programs is the inability to induce an appropriate, protective, immune response. For example, for vaccines against intracellular pathogens cell-mediated immunity, as characterized by cytolytic T-lymphocyte activity, is required4. Such a response can be extremely difficult to elicit, especially by recombinant, soluble protein subunits. This deficiency is due to the inability of these antigens to access the machinery of the appropriate antigen-processing pathway. Following an improved understanding of the mechanisms underlying such processing, as well as the realization that delivery systems can affect, quantitatively and qualitatively, the resulting immune response, the last decade has witnessed an intense research effort in this field4-8. New adjuvant formulations now mostly contain a vehicle that carries antigens to antigen-presenting cells.
Examples of vehicles are generally particulate e.g. emulsions, microparticles, iscoms and liposomes, and microfluidized squalene-in-water emulsions4-8. The main function of such delivery systems is to target associated antigens to antigen presenting cells (APC), including macrophages and dendritic cells. A number of adjuvants that are particulates of defined dimensions (<5 micron) have been shown to be effective in enhancing the immunogenicity of weak antigens in animal models. Two novel adjuvants that possess significant potential for the development of new vaccines include an oil-in-water micro-emulsion and polymeric microparticles.
The parenteral route is still the most common route used for the administration of antigens. However, the induction of an efficient local immune reaction is dependent on the presence of air or food born pathogens at the mucosal surfaces, which presence can result in the production of neutralising antibodies. Furthermore, products given by syringe are inherently more expensive than those which can be taken by mouth or—for example—as a nasal spray. The danger of re-use of needles in underdeveloped countries is a compounding factor.
The tissues of the mucosae encounter the majority of antigens that enter the host and infections of the intestine, respiratory tract and urogenital tract are the most common cause of mortality and morbidity in humans2. With mucosal vaccination it is possible to stimulate both arms of the immune system and provide both humoral (antibody) and cell-mediated responses (cytotoxic lymphocytes)1. Despite the urge for an efficient mucosal vaccine, its introduction is still hindered by the degradation of antigens during transport to and low uptake by the mucosal associated lymphoid tissue (MALT). To circumvent these problems, antigens for mucosal vaccine delivery can be associated to or co-administered with an adjuvant acting simultaneously as efficient delivery system3.9.
Since each part of the MALT has its own specific barriers, each administration route needs its own vaccine delivery system. Oral vaccination is firstly complicated due to degradation of antigens by both the acidic environment in the stomach and the enzymes in the gut. Moreover, the soluble antigens are not always taken up efficiently by the M-cells of the gut associated lymphoid tissue (GALT). By entrapping the antigen in microparticulate adjuvants, the antigen may be protected against degradation on its way to the mucosal tissue and efficiently targeted to and taken up by the M-cells10-13.
Nasal vaccination is mainly complicated by the fast clearance of the antigen and the low uptake by the nasal associated lymphoid tissue (NALT). For antigen transport over the nasal epithelial barrier, three different approaches can be followed: co-administration of the antigen with an adjuvant that contributes to the immune response but is at the same time absorbable by the nasal mucosae, co-administration of the antigen with an absorption enhancer, or entrapment into a microparticulate system to stimulate M-cells, which are also present in NALT, to internalise the antigen14-15.
A number of strategies to produce protective immune responses have in the past been explored. These include:                a) Live attenuated vaccines—a defective pathogen that would be harmless to subjects e.g. nef deleted viruses. These types of vaccines are not safe for use in some cases.        b) Inactivated or ‘killed’ vaccines. These have still not been fully evaluated for their ability to protect against pathogens. For instance, challenge viruses grown in cells matched to the host and vaccine strains may or may not shed their envelope proteins during inactivation. This type of vaccine is illustrated in the development of a more effective rabies virus.        c) Recombinant sub-unit vaccines—or peptide vaccines—seek to stimulate antibodies to the pathogen by mimicking proteins on its surface (e.g. the proposed Hepatitis B vaccine). Sub-unit vaccines researched to date have been strain-specific and have produced poor antibody responses. Recent research into adjuvants has opened up new areas of envelope vaccine research, with some vaccines capable of inducing neutralising antibodies effective against a range of pathogen strains.        d) Recombinant vectored vaccines—incorporate genes or parts of genes of the pathogen into established vaccines using delivery systems. Delivery systems may include live but harmless viruses, such as the canary pox viruses. Vector vaccines have been shown to produce pathogen-specific cytotoxic T cell responses in subjects. These can be enhanced with DNA vaccine priming.        e) DNA vaccines and replicons—involve genetic sequences injected into subjects to induce the expression of antigens by cells. In the case of replicons, these sequences are wrapped in the outer coat of an unrelated virus.        f) Combination vaccines or ‘prime and boost vaccines’. These entail strategies for the combination of two or more different vaccines to broaden or intensify immune responses. Examples include a vector with antigen to prime a T-cell response with a subunit booster to produce antibodies, or delivery of DNA followed by a vector with genes or gene sequences expressing the same gene(s) or gene sequence. It is possible that two different vaccines could be given at the same time, where one acts more rapidly than the other. This would result in a ‘prime-boost’ effect from a single dose.        g) An important recent development in vaccine design is the use of synthetic genes to maximise their expression in the human cell. This technique has been used in the design of HIV vaccines that enhanced immune responses in animals and at least three vaccines using this technique have now entered early stage clinical trials. It is important to realise that evidence of immune responses in subjects do not necessarily mean that the vaccine prevent infection. Prevention of infection has to be confirmed in animal and human trials. The above stated problems associated with vaccines have led to the investigations associated with the present invention.        
The fatty acid/nitrous oxide-based technology comprise of a unique submicron emulsion type formulation within which stable vesicular structures or particles are formed. It was pointed out, inter alia, in WO97/17978 referred to above that nitrous oxide is a natural gas which is also produced synthetically, that it is also known by the trivial name “laughing gas”, and that it has been in use for many years as an inhalation anaesthetic and analgesic, particularly in dentistry.
Nitrous oxide is known to be soluble in water and it has been reported that at 20° C. and 2 atm pressure one liter of the gas dissolves in 1.5 liters of water, see The Merck Index 10th Ed. p. 6499.
There appears to be no suggestion in the literature, other than in the patents and patent applications referred to above, that solutions of nitrous oxide might have any effect on man or animals. As far as the present applicant knows, it has also never been suggested that nitrous oxide may be used in conjunction with fatty acids as an adjuvant to enhance the immune response against antigen-specific diseases.
It is known in the pharmaceutical field that antigens can be formulated in so-called lipid-based formulations. None of these lipid-based formulations are used in combination with nitrous oxide, unlike the present invention in which the combination of nitrous oxide and fatty acids and esters thereof forms the basis of the micro-emulsion adjuvant system. As will be shown below, investigation confirmed the essential role of nitrous oxide in the stimulation of the immune response. The combination of nitrous oxide and fatty acids as an adjuvant for vaccines according to the present invention as described herein shows significant differences to that based on the fatty acids only.